There is a great need for therapies for the treatment of viral diseases. While antiviral drugs such as zidovudine, used in the treatment of human immunodeficiency virus (HIV), and drugs such as ganciclovir, acyclovir, and foscarnet are used in the treatment of herpesvirus infections, significant side effects often limit their effectiveness. The selection and spread of drug-resistant viruses also limits the effectiveness of small molecular weight antiviral drugs. This is a particularly significant problem for drugs targeted against RNA viruses such as HIV, which have a relatively high mutation rate compared to most DNA viruses.
Antiviral vaccines are a viable alternative to postinfection antiviral drug treatments. Ideally, antiviral vaccines protect against primary disease and recurring infections. Efficacy against a particular disease is crucial to the development of a vaccine strategy. Regulatory concerns, particularly related to the safety of vaccines intended for prophylactic use in healthy individuals, must also be considered.
While herpesvirus vaccines have been an active area of both academic and commercial interest, induction of a good, protective immune response in humans has been challenging [R. L. Burke, Current Status of HSV Vaccine Development, in The Human Herpesviruses, 367-379, (B. Roizman, R. J. Whitley and C. Lopez, eds. 1993)]. Live virus vaccines have the risk of establishing latency and reactivating. Live virus vaccines also have the potential of recombining with natural isolates.
Attenuated recombinant viruses and subunit vaccines have been investigated to avoid these risks. Meignier et al describe a recombinant virus resulting from the removal of a region of herpes simplex virus type 1 (HSV-1) required for virulence and the insertion of herpes simplex virus type 2 (HSV-2) glycoprotein genes [J. Infect. Dis., 158:602-614 (1988)]. The viruses had reduced pathogenicity and induced immunity in a number of animal models.
More recently, recombinant herpes simplex viruses with deletions in essential immediate early or early genes have been described. These recombinant viruses are described as being efficacious in inducing immunity and reducing acute replication and establishment of latency of the challenged wild-type virus in mice. Nguyen et al describe replication-defective mutants of HSV-1 that have mutations in the essential genes encoding infected cell protein 8 ("ICP8") or ICP27 [J. Virol. 66:7067-7072 (1992)]. The ICP8 mutant (d301) expresses the products of the .alpha. and .beta. genes while the ICP27 mutant (n504) expresses the products of the .alpha., .beta., and .gamma..sub.1 genes in the cells that the viruses can infect. Both viruses induced antibody responses that were lower than parental (KOS 1.1) virus, but the level induced by the ICP27 mutant was higher than that induced by infection with the ICP8 mutant. Morrison and Knipe later demonstrated that injection of these viruses protected mice against development of encephalitis and keratitis, and decreased the primary replication of virulent challenge virus [J. Virol. 68:689-696 (1994)]. WO95/18852 describes similar replication-defective herpesvirus mutants and WO94/03207 describes vaccines based on these mutants.
Another recombinant virus has been described that has a deletion in the glycoprotein H (gH) coding region [Forrester et al, J. Virol. 66:341-348 (1992); WO92/05263]. This virus forms virions after infection of non-helper cells, but the viruses fail to infect in a subsequent round. Inoculation of mice with the gH deletion virus resulted in a more rapid clearance of the wild-type challenge virus compared to vaccination with chemically-inactivated virus [Farrell et al, J. Virol. 68:927-932 (1994)]. Inoculation of guinea pigs with the gH deleted recombinant virus resulted in reduced primary vaginal disease and reduced recurrences [McLean et al, J. Infect. Dis. 170:1100-1109 (1994)].
Most viruses encode proteinases that function in the processing of viral proteins during infection [W. G. Dougherty and B. L. Semler, Microbiological Reviews, 57:781-822 (1993)]. Biological and biochemical studies have shown that HSV-1 possesses a proteinase that can process another viral protein, the capsid assembly protein (also known as p40, ICP35 and VP22a). Similar proteinases are encoded in the genome of other members of the Herpesviridae. This family of DNA viruses includes HSV-1, HSV-2, human and simian cytomegalovirus (HCMV, SCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus types -6, -7, and -8 (HHV-6, HHV-7, and HHV-8), pseudorabies virus (PRV), bovine herpesvirus (BHV), equine herpesvirus (EHV), and rhinotracheitis virus, among others.
Early work by Preston et al, [J. Virol. 45:1056-1064 (1983)] showed that a temperature-sensitive (ts) mutant in HSV-1 (ts1201) failed to cleave the capsid assembly protein to its lower molecular weight forms at the nonpermissive temperature. This mutant also failed to package viral DNA. By marker rescue, the defect was mapped to a region of the genome in what is now known as the UL26 open reading frame (ORF) [McGeoch et al, J. Gen. Virol. 69:1531-1574 (1988)]. Subsequent analysis showed that two transcripts initiate in the UL26 region, a primary transcript of about 2.1 kb which encodes a protein of 635 amino acids, and a more abundant transcript which is initiated within the UL26 ORF, about 1000 nucleotides 3' of the primary transcript initiation. This smaller transcript encodes a predicted protein of 329 amino acids and is 3' coterminal with the larger 80 kDa ORF encoded by the larger transcript [F. Y. Liu and B. Roizman. J. Virol. 65:206-212 (1991)]. The defect in the ts1201 mutant maps in the 5' region of the longer transcript which has been shown to encode a proteinase activity in HSV-1 [F. Y. Liu and B. Roizman. J. Virol. 65:5149-5156 (1991)] or in simian cytomegalovirus [Welch et al, Proc. Natl. Acad. Sci. U.S.A. 88:10792-10796 (1991)].
Superinfection/transient expression [F. Y. Liu and B. Roizman. J. Virol. 65:5149-5156 (1991)], transient expression [Welch et al, Proc. Natl. Acad. Sci. U.S.A. 88:10792-10796 (1991)], and infection [Preston et al, Virol. 186:87-98 (1992)] studies with the protease domain and the capsid assembly protein domain showed that the proteinase cleaves the capsid assembly protein near its carboxyl terminus. Further studies with the proteins produced in E. coli confirmed that the full-length protein of the UL26 ORF is capable of cleaving itself at two sites as well as cleaving the capsid assembly protein [Deckman et al, J. Virol. 66:7362-7367 (1992)]. DiIanni et al later located the cleavage sites between amino acids 247/248 and 610/611 of the UL26 ORF [J. Biol. Chem. 268:2048-2051 (1993)].
Although the results with ts1201 suggest that the defect in the virus is in its ability to cleave the capsid assembly protein and subsequent encapsidation of DNA, it is not known whether this phenotype is the result of a defect in the protease activity per se, or whether the 5' region of the UL26 ORF encodes some other functions required for capsid assembly and maturation. The processed proteinase domain of the 80 kDa precursor (designated as "VP24" or "N.sub.o ") has been identified in B-capsids [Davison et al, J. Gen. Virol. 73:2709-2713 (1992)] and is retained in A-capsids and C-capsids [F. J. Rixon, Structure and Assembly of Herpesviruses, in Seminars in Virology, vol. 4, 135-144, (A. J. Davison, ed. 1993)] suggesting a structural role for this domain. B-capsids are immature capsids in the nucleus of the infected cell that contain the capsid assembly protein, but not viral DNA. These capsids are thought to be the precursors of A-capsids which fail to package DNA and C-capsids which package DNA with concomitant loss of the capsid assembly protein [B. Roizman and A. Sears, Herpes Simplex Viruses and Their Replication, in Human Herpesviruses, 11-68, (B. Roizman, R. J. Whitley, and C. Lopez, eds. 1993)]. Gao et al constructed and characterized a null mutant virus ("m100") that contains a deletion within the protease domain of the HSV-1 UL26 gene [J. Virol. 68:3702-3712 (1994)]. The mutant virus could be propagated on a complementing cell line but not on noncomplementing Vero cells, indicating that the protease domain of UL26 is essential for viral replication in cell culture. DNA replication occurred at near wild-type levels, but the viral DNA was not processed to unit genome length or encapsidated.
We have generated a recombinant virus to further investigate the role of this domain with respect in vivo effects. The recombinant virus is avirulent in vivo and induces immunity to challenge by wild-type HSV-1.